Human alveolar macrophages do not rely on glucose metabolism upon activation by lipopolysaccharide.

Most macrophages generate energy to mount an inflammatory cytokine response by increased glucose metabolism through intracellular glycolysis. Previous studies have suggested that alveolar macrophages (AMs), which reside in a glucose-poor natural environment, are less capable to utilize glycolysis and instead rely on other substrates to fuel oxidative phosphorylation (OXPHOS) for energy supply. At present, it is not known whether AMs are capable to use glucose metabolism to produce cytokines when other metabolic options are blocked. Here, we studied human AMs retrieved by bronchoalveolar lavage from healthy subjects, and examined their glucose metabolism in response to activation by the gram-negative bacterial component lipopolysaccharide (LPS) ex vivo. The immunological and metabolic responses of AMs were compared to those of cultured blood monocyte-derived macrophages (MDMs) from the same subjects. LPS stimulation enhanced cytokine release by both AMs and MDMs, which was associated with increased lactate release by MDMs (reflecting glycolysis), but not by AMs. In agreement, LPS induced higher mRNA expression of multiple glycolytic regulators in MDMs, but not in AMs. Flux analyses of [13C]-glucose revealed no differences in [13C]-incorporation in glucose metabolism intermediates in AMs. Inhibition of OXPHOS by oligomycin strongly reduced LPS-induced cytokine production by AMs, but not by MDMs. Collectively, these results indicate that human AMs, in contrast to MDMs, do not use glucose metabolism during LPS-induced activation and fully rely on OXPHOS for cytokine production.

The PPAR-γ agonist pioglitazone exerts proinflammatory effects in bronchial epithelial cells during acute Pseudomonas aeruginosa pneumonia.

Pseudomonas aeruginosa is a common respiratory pathogen that causes injurious airway inflammation during acute pneumonia. Peroxisome proliferator-activated receptor (PPAR)-γ is involved in the regulation of metabolic and inflammatory responses in different cell types and synthetic agonists of PPAR-γ exert anti-inflammatory effects on myeloid cells in vitro and in models of inflammation in vivo. We sought to determine the effect of the PPAR-γ agonist pioglitazone on airway inflammation induced by acute P. aeruginosa pneumonia, focusing on bronchial epithelial cells. Mice pretreated with pioglitazone or vehicle (24 and 1 h) were infected with P. aeruginosa via the airways. Pioglitazone treatment was associated with increased expression of chemokine (Cxcl1, Cxcl2, and Ccl20) and cytokine genes (Tnfa, Il6, and Cfs3) in bronchial brushes obtained 6 h after infection. This pro-inflammatory effect was accompanied by increased expression of Hk2 and Pfkfb3 genes encoding rate-limiting enzymes of glycolysis; concurrently, the expression of Sdha, important for maintaining metabolite flux in the tricarboxylic acid cycle, was reduced in bronchial epithelial cells of pioglitazone treated-mice. Pioglitazone inhibited bronchoalveolar inflammatory responses measured in lavage fluid. These results suggest that pioglitazone exerts a selective proinflammatory effect on bronchial epithelial cells during acute P. aeruginosa pneumonia, possibly by enhancing intracellular glycolysis.

HIF-1α Stabilization in Flagellin-Stimulated Human Bronchial Cells Impairs Barrier Function.

The respiratory epithelium provides a first line of defense against pathogens. Hypoxia-inducible factor (HIF)1α is a transcription factor which is stabilized in hypoxic conditions through the inhibition of prolyl-hydroxylase (PHD)2, the enzyme that marks HIF1α for degradation. Here, we studied the impact of HIF1α stabilization on the response of primary human bronchial epithelial (HBE) cells to the bacterial component, flagellin. The treatment of flagellin-stimulated HBE cells with the PHD2 inhibitor IOX2 resulted in strongly increased HIF1α expression. IOX2 enhanced the flagellin-induced expression of the genes encoding the enzymes involved in glycolysis, which was associated with the intracellular accumulation of pyruvate. An untargeted pathway analysis of RNA sequencing data demonstrated the strong inhibitory effects of IOX2 toward key innate immune pathways related to cytokine and mitogen-activated kinase signaling cascades in flagellin-stimulated HBE cells. Likewise, the cell-cell junction organization pathway was amongst the top pathways downregulated by IOX2 in flagellin-stimulated HBE cells, which included the genes encoding claudins and cadherins. This IOX2 effect was corroborated by an impaired barrier function, as measured by dextran permeability. These results provide a first insight into the effects associated with HIF1α stabilization in the respiratory epithelium, suggesting that HIF1α impacts properties that are key to maintaining homeostasis upon stimulation with a relevant bacterial agonist.

Post-treatment with the PPAR-γ agonist pioglitazone inhibits inflammation and bacterial growth during Klebsiella pneumonia.

Agonists of peroxisome proliferator-activated receptor (PPAR)-γ have been suggested as potential adjuvant therapy in bacterial pneumonia because of their capacity to inhibit inflammation and enhance bacterial clearance. Previous studies only assessed the effects of pretreatment with these compounds, thereby bearing less relevance for the clinical scenario. Moreover, PPAR-γ agonists have not been studied in pneumonia caused by Klebsiella pneumoniae, a common human respiratory pathogen of which antibiotic treatment is hampered by increasing antimicrobial resistance. Here we show that administration of the PPAR-γ agonist pioglitazone 6 or 8 h after infection of mice with a highly virulent strain of Klebsiella pneumoniae via the airways results in reduced cytokine and myeloperoxidase levels in the lungs at 24 h after infection, as well as reduced bacterial growth in the lungs and decreased dissemination to distant organs at 42 h post-infection. These results suggest that pioglitazone may be an interesting agent in the treatment of Klebsiella pneumonia.

Integrated single-cell analysis unveils diverging immune features of COVID-19, influenza, and other community-acquired pneumonia.

The exact immunopathophysiology of community-acquired pneumonia (CAP) caused by SARS-CoV-2 (COVID-19) remains clouded by a general lack of relevant disease controls. The scarcity of single-cell investigations in the broader population of patients with CAP renders it difficult to distinguish immune features unique to COVID-19 from the common characteristics of a dysregulated host response to pneumonia. We performed integrated single-cell transcriptomic and proteomic analyses in peripheral blood mononuclear cells from a matched cohort of eight patients with COVID-19, eight patients with CAP caused by Influenza A or other pathogens, and four non-infectious control subjects. Using this balanced, multi-omics approach, we describe shared and diverging transcriptional and phenotypic patterns-including increased levels of type I interferon-stimulated natural killer cells in COVID-19, cytotoxic CD8 T EMRA cells in both COVID-19 and influenza, and distinctive monocyte compositions between all groups-and thereby expand our understanding of the peripheral immune response in different etiologies of pneumonia.

Hypoxia-Inducible Factor-1α in Macrophages, but Not in Neutrophils, Is Important for Host Defense during Klebsiella pneumoniae-Induced Pneumosepsis.

Hypoxia-inducible factor- (HIF-) 1α has been implicated in the ability of cells to adapt to alterations in oxygen levels. Bacterial stimuli can induce HIF1α in immune cells, including those of myeloid origin. We here determined the role of myeloid cell HIF1α in the host response during pneumonia and sepsis caused by the common human pathogen Klebsiella pneumoniae. To this end, we generated mice deficient for HIF1α in myeloid cells (LysM-cre × Hif1α fl/fl) or neutrophils (Mrp8-cre × Hif1α fl/fl) and infected these with Klebsiella pneumoniae via the airways. Myeloid, but not neutrophil, HIF1α-deficient mice had increased bacterial loads in the lungs and distant organs after infection as compared to control mice, pointing at a role for HIF1α in macrophages. Myeloid HIF1α-deficient mice did not show increased bacterial growth after intravenous infection, suggesting that their phenotype during pneumonia was mediated by lung macrophages. Alveolar and lung interstitial macrophages from LysM-cre × Hif1α fl/fl mice produced lower amounts of the immune enhancing cytokine tumor necrosis factor upon stimulation with Klebsiella, while their capacity to phagocytose or to produce reactive oxygen species was unaltered. Alveolar macrophages did not upregulate glycolysis in response to lipopolysaccharide, irrespective of HIF1α presence. These data suggest a role for HIF1α expressed in lung macrophages in protective innate immunity during pneumonia caused by a common bacterial pathogen.

Adherence Affects Monocyte Innate Immune Function and Metabolic Reprogramming after Lipopolysaccharide Stimulation In Vitro.

Circulating nonadherent monocytes can migrate to extravascular sites by a process that involves adherence. Alterations in intracellular metabolism shape the immunological phenotype of phagocytes upon activation. To determine the effect of adherence on their metabolic and functional response human monocytes were stimulated with LPS under nonadherent and adherent conditions. Adherent monocytes (relative to nonadherent monocytes) produced less TNF and IL-1ß (proinflammatory) and more IL-10 (anti-inflammatory) upon LPS stimulation and had an increased capacity to phagocytose and produce reactive oxygen species. RNA sequencing analysis confirmed that adherence modified the LPS-induced response of monocytes, reducing expression of proinflammatory genes involved in TLR signaling and increasing induction of genes involved in pathogen elimination. Adherence resulted in an increased glycolytic response as indicated by lactate release, gene set enrichment, and [13C]-glucose flux analysis. To determine the role of glycolysis in LPS-induced immune responses, this pathway was inhibited by glucose deprivation or the glucose analogue 2-deoxy-d-glucose (2DG). Although both interventions equally inhibited glycolysis, only 2DG influenced monocyte functions, inhibiting expression of genes involved in TLR signaling and pathogen elimination, as well as cytokine release. 2DG, but not glucose deprivation, reduced expression of genes involved in oxidative phosphorylation. Inhibition of oxidative phosphorylation affected TNF and IL-10 release in a similar way as 2DG. Collectively, these data suggest that adherence may modify the metabolic and immunological profile of monocytes and that inhibition of glycolysis and oxidative phosphorylation, but not inhibition of glycolysis alone, has a profound effect on immune functions of monocytes exposed to LPS.

Interleukin-33 improves local immunity during Gram-negative pneumonia by a combined effect on neutrophils and inflammatory monocytes.

Pneumonia represents a major health care burden and Gram-negative bacteria provide an increasing therapeutic challenge at least in part through the emergence of multidrug-resistant strains. IL-33 is a multifunctional cytokine belonging to the IL-1 family that can affect many different cell types. We sought here to determine the effect of recombinant IL-33 on the host response during murine pneumonia caused by the common Gram-negative pathogen Klebsiella pneumoniae. IL-33 pretreatment prolonged survival for more than 1 day during lethal airway infection and decreased bacterial loads at the primary site of infection and distant organs. Postponed treatment with IL-33 (3 h) also reduced bacterial growth and dissemination. IL-33-mediated protection was not observed in mice deficient for the IL-33 receptor component IL-1 receptor-like 1. IL-33 induced a brisk type 2 response, characterized by recruitment of type 2 innate lymphoid cells to the lungs and enhanced release of IL-5 and IL-13. However, neither absence of innate lymphoid cells or IL-13, nor blocking of IL-5 impacted on IL-33 effects in mice infected with Klebsiella. Likewise, IL-33 remained effective in reducing bacterial loads in mice lacking B, T, and natural killer T cells. Experiments using antibody-mediated cell depletion indicated that neutrophils and inflammatory monocytes were of importance for antibacterial defense. The capacity of IL-33 to restrict bacterial growth in the lungs was strongly reduced in mice depleted of both neutrophils and inflammatory monocytes, but not in mice selectively depleted of either one of these cell types. These results suggest that IL-33 boosts host defense during bacterial pneumonia by a combined effect on neutrophils and inflammatory monocytes. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Complement factor C5 inhibition reduces type 2 responses without affecting group 2 innate lymphoid cells in a house dust mite induced murine asthma model.

BACKGROUND: Complement factor C5 can either aggravate or attenuate the T-helper type 2 (TH2) immune response and airway hyperresponsiveness (AHR) in murine models of allergic asthma. The effect of C5 during the effector phase of allergen-induced asthma is ill-defined. OBJECTIVES: We aimed to determine the effect of C5 blockade during the effector phase on the pulmonary TH2 response and AHR in a house dust mite (HDM) driven murine asthma model. METHODS: BALB/c mice were sensitized and challenged repeatedly with HDM via the airways to induce allergic lung inflammation. Sensitized mice received twice weekly injections with a blocking anti-C5 or control antibody 24 h before the first challenge. RESULTS: HDM challenge in sensitized mice resulted in elevated C5a levels in bronchoalveolar lavage fluid. Anti-C5 administered to sensitized mice prior to the first HDM challenge prevented this rise in C5a, but did not influence the influx of eosinophils or neutrophils. While anti-C5 did not impact the recruitment of CD4 T cells upon HDM challenge, it reduced the proportion of TH2 cells recruited to the airways, attenuated IL-4 release by regional lymph nodes restimulated with HDM ex vivo and mitigated the plasma IgE response. Anti-C5 did not affect innate lymphoid cell (ILC) proliferation or group 2 ILC (ILC2) differentiation. Anti-C5 attenuated HDM induced AHR in the absence of an effect on lung histopathology, mucus production or vascular leak. CONCLUSIONS: Generation of C5a during the effector phase of HDM induced allergic lung inflammation contributes to TH2 cell differentiation and AHR without impacting ILC2 cells.